The aldehyde form of vitamin A, retinal, is the ligand of the visual pigments in both rod and cone photoreceptors. This ligand can either act as an inverse agonist, placing the protein into its inactive conformation, or an agonist, moving the protein into an active conformation and activating the visual transduction process. This small molecule is thus critical to the visual process. Our research is focused on the study of the interaction of this ligand with the various rod and cone opsins and the role of one protein, RPE65, which is critical for the generation of the form of retinal that binds to those opsins, the 11-cis isomer. Analogues of retinal will bind to the opsins and can be used to manipulate these G-protein receptors. Using a variety of physiological techniques, we propose to use retinal analogues to address a number of hypotheses particularly focused on studies of the green rod and three cone opsins of the salamander. With these structurally modified forms of retinal, the protein conformation can be altered such that particular steps in the ligand binding or the protein activation can be examined. Through these studies, we are addressing hypotheses that the rod and cone opsins have very different interactions with 11-cis-retinal and that their mechanisms for the release of the end product, all-trans-retinol, also differs. The protein RPE65 is critical for the generation of 11-cis-retinal, although the exact function of this protein is still unknown. We are testing the hypothesis that the protein is a scaffolding protein, involved in a complex that brings about the isomerization/oxidation of all-trans-retinol to 11-cis-retinal. Using a cell line HEK296, which itself contains RPE65, and an opsin expression system which then provides a "trap" for the 11-cis-retinal, we propose to determine which other retinoid-binding/metabolizing proteins are critical for this process. Secondly, using animal models, which have had one or more of the retinoid-binding proteins knocked out, we will examine the levels of RPE65 and the ability of the animal to produce 11-cis-retinal. It is now being clearly shown that many retinal disorders are due to errors in retinoid metabolism or binding of the ligand with the visual pigments. Examples are various: Stargardt's disease, autosomal recessive retinitis pigmentosa, and Leber's congenital amaurosis. This project is focused on determining the mechanisms of retinoid metabolism and retinal-opsin protein interactions so that a rational design of prevention or therapies for these blinding disorders can be undertaken.